In vitro, ex vivo and in vivo experimental results speak to the effective use of certain Pt(IV) compounds in the treatment of viral infections and their associated pathologies. Use of these compounds may also provide for recovery of immunological suppression by viruses, and be useful for the development of vaccines. This application is relevant to retroviruses, oncoretroviruses, filoviruses, arenaviruses and other viruses expressing nucleocapsid proteins or zinc-binding proteins where a Pt(IV) compound interacts with zinc-binding domains or zinc-binding sites, altering activities of the corresponding proteins. Other applications include interactions with the CCCH-zinc finger protein family, MCPIP-1, -2, -3, and -4, relevant to macrophage inflammatory responses (Liang et al., 2008) and adipogenesis (Younce et al., 2009). Pt(IV) compounds are proposed to also be relevant to the specific inhibition of other proteins containing cysteine-rich regions that could coordinate metals. The HIV-1 Trans-Activator of Transcription (Tat) protein, for instance, contains such a region (Huang and Wang, 1996; Frankel et al., 1988).
HIV-1 and other lentiviruses deliver two copies of single-stranded RNA into host cells where these are reverse-transcribed to DNA (deoxyribonucleic acid), incorporated into host DNA (“proviral DNA”), transcribed to make viral proteins, proviral circular (c)DNA and viral RNA (ribonucleic acid), then transported and packaged into virions. Gag protein is transcribed as a single structural polyprotein, and then sequentially cleaved to generate its components. A ribosomal frameshift may generate another viral protein, Gag-Pol, providing the viral enzymes: protease, reverse transcriptase and integrase. For HIV-1, protease cleaves Gag into three final nucleocapsid proteins (NCp): NCp15, NCp9 and NCp7.
Current interest in Gag owes partly to its ability to independently form virus-like particles (VLPs) in mammalian cells; in vitro, this occurs in the presence of nucleic acids and a cofactor (Campbell and Rein, 1999; Campbell, 2001). Immature VLPs do not include a surrounding outer membrane, yet emphasize the thermodynamic role of Gag proteins associated with RNA. Presence of nucleic acids is critical for Gag VLP production although Gag demonstrates preferential selectivity for viral RNA over other available oligonucleotides (Berkowitz, 1996).
The Gag-RNA interaction is proposed to rely upon unique zinc finger domains found in the NCp7 product of Gag. While zinc finger domains are frequent to the human genome—4500 CCHH zinc finger domains in 564 proteins—the CCHC zinc finger domain specific to all known nucleocapsid proteins of retroviruses is found in only 17 zinc domains of 9 human proteins. HIV-1 nucleocapsid protein 7 (NCp7) contains two such domains. Furthermore, these zinc finger domains of Cys-XX-Cys-XXXX-His-XXXX-Cys (or CCHC, where C/Cys is cysteine; H/His is histidine; and X is a variable residue) include 15 basic residues and two aromatic residues (phenylalanine and tryptophan)—each strategically located adjacent to the N-terminus Cys in each loop (see sequences below). These aromatic residues have special significance in their association with unpaired nucleotides through pi stacking; tryptophan (W37) associates between adjacent cytosine and guanine nucleotides, stacking with guanine (Morellet, 1998). Zinc ions (Zn2+) coordinate to each of the 3 Cys (through the sulfur atoms) and one His residue (through the nitrogen atoms) in each loop. Cys49 and its sulfur have experimentally been found to be the most reactive sites (Maynard and Covell, 2001) and are expected to be the first event of zinc destabilization from the loop. NCp7 zinc-binding domains participate in essential viral conformational RNA strand changes known as “chaperoning”—both prior to virion packaging (strand aggregation) and prior to reverse transcription (helix destablilzation). Omission of either NCp7 zinc domain results in unexpected premature viral DNA synthesis in virus producer cells and the production of noninfectious particles with a high level of viral DNA, as opposed to RNA (Houzet, 2008). Synchronization of cellular translational events is also regulated through NCp7, making the nucleocapsid protein a promising target for therapeutic treatments (Didierlaurent, 2008).
Primary sequences of NCp7 highlighting residue properties and zinc coordination sites:
SEQ ID NO 2:IQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCT ERQANFLGKIWPSHKGRPGNF (Cruceanu, 2006)
SEQ ID NO 3:MQRGNFRNQRKNVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCT ERQANFLGKIWPSYKGRPGNFL (Anzellotti, 2006)
Multiple and cell-specific activities act through NCp7, emphasizing the efficient redundancy notorious to viruses. Besides serving as structural housing components of genomic viral RNA (the nucleocapsid), NCp7 proteins are required for genomic RNA packaging and chaperoning; transfer RNA annealing; integrase-mediated strand-transfer; virus assembly and budding; Gag trafficking and processing; and temporal control of reverse transcription (Didierlaurent, 2008; Houzet, 2008; Thomas and Gorelick, 2008; Mascarenhas and Musier-Forsyth, 2009). While other peptides and proteins may substitute for these activities—resulting in cell-specific effects—optimized efficiencies by NCp7 may be critical to successful virus infection and production. Additionally, zinc finger domains of NCp7 are integral to the full length Gag protein prior to each of three sets of protease cleavages resulting in the final products: matrix (NCp15), capsid (NCp9), nucleocapsid (NCp7) and p6. Therefore, zinc-finger domain alterations by coordinating to platinum(IV) compounds may range across effects of full length Gag precursor protein to the fully cleaved mature nucleocapsid protein.
Alterations to the zinc-finger domain configuration result in profound changes to its protein function. Site-directed mutagenesis of HIV-1 NCp7, substituting leucine for proline-31 in the peptide linker between its two zinc-binding domains, resulted in the formation of non-infectious and immature viral particles (Morellet, 1994). Even subtle residue substitutions (interchanging cysteines and histidines) in the zinc-binding domain altered the cooperativity of DNA helix-coil transitions such that the protein lost its nucleic acid-chaperoning capacity (Guo, 2002; Williams, 2002; Ramboarina, 2004); a mutant form of nucleocapsid with both CCHC domains changed to SSHS is unable to bind zinc and is impaired to facilitate minus-strand transfer in reverse transcription (Guo, 2000). Other mutations to this domain result in premature reverse transcription and unusual incorporation of viral DNA into virion progeny (Thomas, 2008). Mutations in the N-terminal domain of the nucleocapsid protein result in a strong reductions of proviral DNA in host cells (Berthoux, 1997; Tanchou, 1998). Numerous other investigators report that mutations to the NC protein, as part of Gag and/or as mature NCp7, result in production of viral particles defective in replication (Dannull, 1994; Ottmann, 1995; Poon, 1996; Schmalzbauer, 1996) and/or defective in genomic RNA packaging (Aldovini and Young, 1990; Gorelick, 1990; Dorfman, 1993; Mizuno, 1996).
Manrique et al. (2004) addressed the role of the feline immunodeficiency virus (FIV) nucleocapsid (NC) protein in late stage virus replication. Defective phenotypes with respect to particle formation and RNA binding were observed when the first cysteine residue in the NC proximal zinc finger, or when basic residues connecting both zinc fingers, were replaced. HIV-1, SIV and FIV each share the highly conserved CX2CX4HX4C sequence of NC zinc finger domains (Table 1; Thomas and Gorelick, 2008). The function of FIV Gag proteins parallel those of HIV-1 Gag, as reviewed by Luttge and Freed (2010), including short peptide motifs essential for proper release of assembled virions from infected cells, RNA chaperoning, trafficking to membranes and virus assembly. Similarities in structure and functions of Gag/nucleocapsid zinc finger domains, together with in vitro HIV-1 data illustrating associated defective particle phenotypes and reductions in virion production support the success of human translational efficacies.
TABLE 1Comparative Sequences of Nucleocapsid Zinc DomainsN-TerminalC-TerminalBasicAcidVirus ProteinSizeZinc fingerZinc finger ResiduesResiduesHIV-1p755SEQ ID NO 4:SEQ ID NO 7: 15 (27%)4 (7.3%)CFNCGKEGHIAKNC CWKCGKEGHQMKDCSIVp852SEQ ID NO 5:SEQ ID NO 8: 12 (23%) 1 (1.9%)CWNCGKEGHSARWC CWKCGQMGHVMAKCFIVp1066SEQ ID NO 6:SEQ ID NO 9: 14 (21%)1 (1.5%)CFNCKKPGHLARQCCNKCGKPGHVAAKCHIV-1 sequence data from GenBank accession number AF324493 (Ottmann, 1995).SIV sequence data from GenBank accession number AY817672 (Gorelick, 1999).FIV sequence data from GenBank accession number M25381 (Manrique, 2004).
In a pilot study of two FIV-infected felines, a short increase in peripheral blood mononuclear cell proviral load was observed in both animals immediately following (14 days later) the final dosing of the platinum(IV) compound, FX101. Proviral loads declined over subsequent months, even below pretreatment baseline measures, together with undetectable plasma viremia. The initial spike in proviral load following drug clearance may reflect a temporary yet insufficient recovery of NCp7 in its role as helix destabilizer, preceding incorporation into host DNA. Other studies (in vitro p24, in vivo plasma viremia and in vitro reduced virion production) may reflect inhibition of NCp7 in its role as strand aggregator preceding viral assembly. Ultimately, a long term in vivo reduction of both proviral DNA burden and plasma viremia support a virus-debilitating target with corresponding host relief from daily dosing.
TABLE 2Alignment of Group M HIV-1 Nucleocapsid Protein Sequences.N-terminalC-terminalZinc FingerZinc FingerM-Group SEQ ID NO 10:SEQ ID NO: 14ConsensusCFNCGKEGHIARNCCWKCGKEGHQMKDCA1SEQ ID NO 11:SEQ ID NO: 14---------L------------------A2SEQ ID NO 11:SEQ ID NO: 14---------L------------------BSEQ ID NO 12:SEQ ID NO: 14-----------K----------------CSEQ ID NO: 10SEQ ID NO: 14----------------------------DSEQ ID NO: 12SEQ ID NO: 14-----------K----------------F1SEQ ID NO: 12SEQ ID NO: 15 -----------K-------R-------GSEQ ID NO: 11SEQ ID NO: 14---------L------------------HSEQ ID NO: 10SEQ ID NO: 15-------------------R-------KSEQ ID NO: 10SEQ ID NO: 14----------------------------01-AESEQ ID NO: 11SEQ ID NO: 14---------L------------------02-AGSEQ ID NO: 11SEQ ID NO: 14---------L------------------03-ABSEQ ID NO: 13SEQ ID NO: 14------D--L------------------04-CPXSEQ ID NO: 11SEQ ID NO: 14---------L------------------06-CPXSEQ ID NO: 11SEQ ID NO: 14---------L------------------07-BCSEQ ID NO: 10SEQ ID NO: 14----------------------------08-BCSEQ ID NO: 12SEQ ID NO: 14-----------K----------------10-CDSEQ ID NO: 12SEQ ID NO: 15-----------K-------R-------11-CPXSEQ ID NO: 11SEQ ID NO: 14---------L------------------12-BFSEQ ID NO: 12SEQ ID NO: 15 -----------K-------R-------14-BGSEQ ID NO: 11SEQ ID NO: 14---------L------------------NL4-3SEQ ID NO: 12SEQ ID NO: 15-----------K----------------
Applications across most lentivirus strains and subtypes are anticipated since the zinc binding domain is highly conserved. Consensus M-Group HIV-1 Subtypes, for example, are summarized in Table 2 from Thomas and Gorelick (2008), using the Los Alamos HIV Sequence Database. Since the NC zinc finger is conserved across HIV-1, HIV-2, SIV (Morellet, 2006) and FIV (Manrique, 2004), activities across these mutants are anticipated.
Positively-charged NCp7 is well-suited to accommodate the negatively-charged phosphate groups of polynucleotides, positioning base complements within range to form energetically-stabilizing hydrogen bonds. These effects would be especially favorable in the context of a lipid membrane (hydrophobic), and could possibly include diffusional forces generated by changing solubility dynamics as folding transpires. It has been suggested that the rapid conformational changes of NCp7 required to facilitate observed interactions with both ssDNA and dsDNA (single stranded and double stranded; analogous to RNA) could involve alternating pi-stacking interactions of key aromatic residues with DNA nucleotides (Cruceanu, 2006). Aromatic residues reduce the HOMO-LUMO (highest occupied molecular orbital—lowest unoccupied molecular orbital) energy gap in pi-stacking groups, enhancing these associations (Ishida, 1990). Hence, these aromatic residues could essentially act as switches. Any alterations to their properties would have profound effects on Gag-RNA interactions. Interestingly, the precursor to NCp7, NCp9, contains a length of hydrophobic residues and demonstrates significantly slower DNA interactions (Khan and Giedroc, 1994). Noncovalent pi stacking interactions between proline and tryptophan residues (˜7 kcal/mol, comparable to hydrogen bonding) have recently been measured, despite proline's lack of aromaticity (Biedermannova, 2008), adding another possible residue contributing to such interactions.
Zinc is critical to the functionality of nucleocapsid proteins; platinum, however, can coordinate in these metal-binding peptides. Transplatin has been shown to displace zinc (Burdette, 2006), owing to the geometry of cysteine residue coordination sites, with displacement of Pt(II)-coordinated chloride ions being an early step (Ivanov, 1998). Two Pt(II) equivalents may be required to fully eject zinc, although there are several observations where metal coordination sharing a single chloride occurs (—Pt—Cl—Zn—); importantly, these studies have been limited to Pt(II) structures. Using an aromatic ligand to direct trans-Pt(II) complexes toward the tryptophan (W37) of NCp7 zinc fingers, trans-[Pt(II)Cl(bispyridine)(9-ethylguanine)] eventually displaces zinc, transitioning through a Pt—S—Zn bridging complex with the loss of chloride ions (Anzellotti, 2006). Gold and palladium have each also demonstrated the capacity to form sulfide-bridging interactions with zinc-like domains (de Paula, 2009), suggesting that alternative modes may contribute additional interactions with the zinc finger domain. Previous small molecule approaches to zinc finger domain interruptions include electrophilic attack by nitrosylated organic aromatics, disulfides, disulfoxides, etc., although these are both nonspecific (toxic) and unstable (Rice, 1997). The nature of specific metal uptake by cells may provide improved specificity for this approach, concentrating metal species within membrane compartments shared by viruses during both infection and assembly.
Current antiviral therapeutics require daily dosing with associated toxicities and development of drug-resistance. Our studies suggest a relief from daily dosing capable of maintaining low viral loads, both in viremia and proviral load, over successive months. It is proposed that viral progeny incorporate the small molecule into their nucleocapsid proteins, thus impairing successful replication in subsequent host cells. Further, alterations to zinc fingers of nucleocapsid proteins have been shown to result in vast differences in viral RNA/DNA splicing and packaging, impacting subsequent infection viral production pathways (Houzet, 2006). Inhibitory effects would then be compounded with first cycle effects, amplifying activities. Mass spectrometric analysis confirms that select Pt(IV) compounds target CCHC nucleocapsid zinc domains of lentiviral Gag proteins, thus accounting for altered activities known to require zinc in the active site. Effects include inhibition of processes such as mature virion formation, of proviral DNA incorporation and of Gag polyprotein processing.
Both selenite (SeO3) and ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) interfere with zinc finger-containing DNA binding domains (Larabee, 2002), albeit via distinct interactions. While both of these selenium compounds successfully ejected the zinc atom from the transcription factor Sp1 DNA-binding domain, ebelsen induced the formation of a disulfide bond (oxidation) in the vacated protein while selenite also produced a sulfur-coordinated selenium species with the two available cysteines (Larabee, 2009). Thus, different modes of action were ultimately effective in achieving inhibition of DNA binding through alteration of the zinc finger by selenium.
Other metals shown to compete or coordinate with zinc finger domains include arsenite (Ding, 2009); cobalt(III) (Harney, 2009); platinum(II), palladium(II) and gold (III) (de Paula, 2009); cadmium(II) and mercury(II) (Heinz, 2009).
Other Factors
Trans, cis-[Pt(en)(OH)2Cl2] and trans, cis-[Pt(en)(OH)2I2] exhibited different reactions to albumin proteins. The difference is due to iodo attack of the cysteine residue, resulting in a sulfenic acid derivative. Low molecular weight thiols react with Pt(IV)-Iodo bonds of trans, cis-[Pt(en)(OH)2I2] forming chelate-ring-opened Pt(II)(en) species. (Kratochwil and Bednarski, 1999). Given the high proportion of thiols in zinc fingers, these types of interactions may be significant. It also suggests that Pt(IV)iodo compounds may prove unexpectedly effective.
Pt(IV) compounds have been reported to have biological actions distinct from their Pt(II) counterparts, even when they show cross-resistance (Hamberger, 2009). The octahedral geometries of Pt(IV) complexes are unusually substitution and kinetically inert; this stability can be explained by its filled 5dt2 low spin configuration coordinated with weak field ligands resistant to oxidation. Pt(IV) may require reduction to the Pt(II) species to become cytotoxic (Bednarski, 2007; Mellor, 2005; Galanski, 2003); several Pt(IV) compounds have been reported stable against biological reductants such as glutathione (Kratochwil and Bednarski, 1999). DNA alkylation by cisplatin is proposed to transition through the exchange of a chloride with an aquo ligand, leading to a positively-charged Pt(II) intermediate that approaches negatively-enveloped DNA. In contrast, nucleocapsid proteins are highly basic. A positively charged Pt species analogous to those alkylating DNA would be electronically hindered from interaction with a basic protein such as NCp7; being substitution inert may be a critical feature of the Pt(IV) capacity to interact with the zinc coordination site.
The absence of myelosuppression from our in vivo studies using Pt(IV) compounds may reflect cytotoxicities limited to specific transformed cell lines rather than to primary cells. Other Pt(IV) compounds have been reported to exhibit direct cytotoxicities in rat and human astrocyte cell lines, without affecting the viability of rat primary astrocytes, nor their functions (Markovic, 2005); in contrast, cisplatin was toxic to both primary cells and transformed cell lines in these studies. While cisplatin can be used to treat certain tumors in the dog, it cannot be utilized in the cat because of fulminant pulmonary oedema that occurs at standard doses (Barabas, 2008); yet, we have successfully administered repeated doses of a Pt(IV) compound to cats without complications. Clinical chemistry panels, complete blood counts and urinalysis from FIV studies in cats evidence no indications of adverse reactions to this therapeutic when administered twice weekly over 4 weeks at the proposed therapeutic dose (6-8 mg/dose intravenous).
While alkylation of peripheral blood leukocyte DNA occurs with cisplatin and its analogs, neither impairment of leukocyte function nor myelosuppression have been observed in our studies. Other reports of Pt(IV) compounds, such as those produced by hydrogen peroxide oxidation, are inactive as antitumor agents and have been shown incapable of unwinding PM2-DNA (Peritz, 1990). Experiments distinguish between Pt(IV) and Pt(II) compounds, both of which exhibit cytotoxicity in ovarian cell lines (A2780, A2780/C30, and CHO), yet only the Pt(II) compounds show evidence of covalent binding to single- or double-stranded DNA by highly sensitive nuclear magnetic resonance (NMR) detection studies (Bose, 2008). Further, some Pt(IV) compounds do not accumulate as cisplatin analogs do (New, 2009). These observations and measures “represent a clear paradigm shift not only in expanding the molecular targets for Pt . . . but also in strategic development . . . ” (Bose, 2008). Some Pt(IV) compounds resistant to reduction have failed to react with DNA in the presence of either low (0.015 mM) or high (3.0 mM) concentrations of glutathione (Kratochwil and Bednarski, 1999), hence interactions exclusive of DNA interactions are evidenced for platinum compounds of the higher oxidation state (i.e., +4). The metallo-biochemistry of certain platinum(IV) compounds focuses on cellular and molecular events beyond DNA binding (Bose, 2002).
Standard in vitro HIV antiretroviral testing has focused upon viral targets such as inhibitors of transcription, infection, viral protease and integrase. The targets for certain platinum(IV) compounds, however, are the zinc finger domains of nucleocapsid proteins, whose activities may not be accurately reflected by these test methods. For instance, deletion of both zinc fingers causes activation of reverse transcription in virus producer cells (Didierlaurent, 2008), which would appear to be counteractive as an antiviral if viewed as an inhibitor of transcription. Furthermore, depending on the specific mutation of the zinc finger motif, increases (rather than decreases) of infectivity have been reported (Mark-Danieli, 2005), although these mutations coincided with genomic encapsidation of larger amounts of foreign RNA in the production of “normal” virion-like particles. Changes to viral transcription, protease and integrase activities may be indirect consequences of the primary mechanism of action—alterations in viral RNA/DNA “chaperoning”, Gag processing and virion assembly due to alterations of the zinc domain.
Most available HIV therapeutics belong to classes of nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-NRTI's or protease Inhibitors (PIs). While no maturation inhibitors have yet achieved FDA approval, this new class of therapeutics includes both Bevirimat (PA-457 or MPC-4326; CS Adamson, 2009) and Vivecon (MPC-9055), both by Myriad Pharmaceuticals and in clinical trials. Azodicarbonamide (ADA) had also proceeded into clinical trials. The utility of maturation-inhibiting antiretrovirals recognizes the fundamental roles of highly conserved Gag viral domains, applicable across multiple strains.
With respect to Pt(IV) compounds of octahedral geometries, it seems interesting that trans-halide geometries have demonstrated antiviral activities while corresponding cis-halide geometries have been ineffective. Pt(IV)s of octahedral geometries form many thermally stable and kinetically inert complexes (Cotton and Wilkinson, 1972, p. 1040), reducing toxic side effects associated with Pt(II)'s. It may have been fortunate to have first examined virus inhibition using feline T-cells, since localization of Gag proteins differs between T-cells and macrophages—the former localize to plasma membranes while the latter distribute to multivesicular bodies (Joshi, 2009). Gag protein associations with cholesterol have been identified for macrophages in multivesicular bodies (Lindwasser and Resh, 2004); cholesterol is well known to traffic with caveolin proteins, so this could offer alternative interactions since Pt(IV)s appear to partition to caveolar fractions as well as to lipid rafts.
A number of gamma-retroviruses have been identified to contain the classical caveolin-1 binding domain (CBD) motif rich in aromatic residues and exhibiting the characteristic spacing φxxxxφxxφ or φxφxxxxφ; φ=W, F, Y (Yu, 2006). Other interactions between viruses and caveolin-1 proteins may occur through nonclassical motifs, and have been observed to involve the cytoplasmic tail of caveolin-1. Since precipitation of caveolar proteins from sucrose-gradient extractions included a high proportion of Pt(IV) distribution to that fraction, direct associations between the protein and Pt are implied. Whether these associations contribute to nucleocapsid protein inhibitions is yet to be explored. Nevertheless, Pt specifically distributed to the most buoyant lipid raft fraction as well, creating additional modes of exposure to sites of viral assembly. Targeting to lipid rafts may prove advantageous to certain antiviral applications.
Matrix Metalloproteinases
It is noteworthy that we observed marked reductions in both MMP-2 and MMP-9 gelatinases throughout our studies. Matrix metalloproteins (MMP's) are characterized by zinc-binding domains also, although their chelating residues differ from those of lentiviral nucleocapsid proteins. The interaction of Pt(IV) compounds with these zinc domains offers therapeutic value; inhibition of the zinc domain of MMP-3 has been reported for certain Pt(II) compounds comprising 3 labile ligands (Arnesano, 2009).
MMPs are a large family (26 at present) of zinc-dependent proteases with multiple roles in extracellular matrix remodeling (Amalinei, 2007), signaling pathway regulations, cell morphology, immune (Elkington, 2009) and inflammatory responses (Manicone and McGuire, 2008) and in the release of soluble factors (Van Lint and Libert, 2007). Most MMPs are secreted in a latent (pro-MMP) form and activated either by proteolytic or oxidative disruption of a conserved cysteine switch (PRCGVPDLGR). Tissue inhibitors of metalloproteinases (TIMPs) physiologically regulate MMPs. Interestingly, this regulation involves interactions between cysteines on TIMPs and zinc metal (Zucker, 1998), otherwise coordinated to histidines and gluatamic acid residues within the MMP catalytic site. Involvement of MMPs in neurodegenerative diseases, oncological disorders, autoimmune and cardiovascular and other diseases identifies them as therapeutic targets.
Data from in vivo studies of mice and cats demonstrate significant reductions in plasma MMPs with administration of certain Pt(IV) compounds. Both MMP-2 and MMP-9 levels were reduced, although no corresponding changes to measured TIMPs accounted for these reductions. MMP's are reported to be particularly involved in the pathogenesis of HIV infections (Webster and Crowe, 2006). These data suggest an interaction between the catalytic zinc binding site and platinum complexes that may contribute inhibitory effects observed for virus production and infiltration. Importantly, platinum coordination to cysteine residues on TIMPs might affect TIMP regulation of MMPs. The MMP's coordinate Zn mainly through N (of His) or O (of Glu or Asp) atoms (Tallant, 2009), different from the mostly S(Cys)-chelated Zn in Gag proteins.
The core domain of MT1-MMP (aka MMP-14, membrane bound) contains four highly occupied metal sites binding two zinc and two calcium ions in a similar manner to the classical MMPs, but lacking a third calcium binding site. The catalytic zinc is chelated by nitrogens on 3 histidines and one cysteine. The second ‘structural zinc’, ZN002, is similarly coordinated to nitrogens of 3 histidines and one carboxylate oxygen of aspartate. MT1-MMP is found in cholesterol-rich lipid rafts on the plasma membrane. Upregulation of caveolin-1 has also been related to the inhibition of MT1-MMP through its surface regulation (Kim and Chung, 2008).
Hepatitus C and Other Viruses
The nonstructural protein 5A (NS5A) of HCV is essential to viral RNA replication and appears to modulate the host cell environment (Marcotrigiano and Tellinghuisen, 2009). NS5A is a large hydrophilic phosphoprotein with three domains; the amino-terminal domain notably coordinates a single zinc atom to 4 cysteines (Cys39, Cys57, Cys59 and Cys80) and is required for virus replication, interferon resistance and apoptosis (Liang, 2007). NS5A is an active component of HCV replicase, a key regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth; the amphipathic-helix at its amino terminus contains anchors to the host cell membrane (Moradpour, 2005). Mutations of NS5A disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication (Moradpour, 2005). Conserved among the Hepacivirus and Pestivirus genera, the zinc-coordinating cysteines follow the pattern of CX17CXCX20C. Mutation of any of the four cysteine residues coordinating the NS5A zinc atom produces a lethal phenotype for HCV RNA replication, suggesting this motif is absolutely required for functionality (Tellinghuisen, 2004).
Another HCV nonstructural 3 (NS3) protein, a serine proteinase, is zinc-containing, whose distinct auto-proteolytic and protease activities are both zinc-dependent, with an absolute requirement for cysteine residues 1123, 1125 and 1171 within NS3 (Tedbury and Harris, 2007). Spectroscopic studies have shown that changes in the conformation of the zinc-binding site correlate with changes in the specific activity of the enzyme, such that the NS3 proteinase is inhibited by compounds capable of extracting zinc from its native coordination sphere (De Francesco, 1999). NS3 is also required for virus maturation (De Francesco, 1999). Pt(IV) compounds may interact with these key HCV viral proteins—NS5A and/or NS3—preventing viral activities or virus maturation, and providing therapeutic value.
Junin virus, a South American arenavirus, is the aetiological agent of Argentine haemorrhagic fever. A zinc-coordination site of the carboxy-terminal region of its N protein corresponds to the sequence CX2HX23CX4C. Specificity for zinc binding was demonstrated by competition with other divalent metal ions (Tortorici, 2001). Inactivation of the arenavirus by antiretroviral zinc finger-reactive compounds resulted in particles which retained the immunoreactivity of their surface glycoproteins, providing a potential antigenic agent for the development of a vaccine (Garcia, 2009) or development of an immunological adaptive response. Production of inactive or dysfunctional virions in such a manner may extend to numerous vaccine applications; studies evaluating such potentials are in progress.
Examples of Viruses Containing Zinc-Binding Domains
Hantaviruses display a highly conserved CCHC-type classical zinc finger domain that participates in viral assembly and host-pathogen interactions (Estrada, 2009).
The nucleocapsid protein VP30 of Marburg and Ebola fiolviruses each contain a zinc-binding motif (Enterlein, 2006).
Human T-Cell Lymphotropic Virus Type 1 is a retrovirus containing two CCHC zinc finger domains in its nucleocapsid domain; infection can lead to adult T-cell leukemia or demyelinating diseases. As compared with HIV-1, HTLV-1 shares nucleic acid chaperoning and packaging activities, although exhibiting some differences in these overall efficiencies (Darugar, 2008)
Herpes Simplex Virus (HSV) replication requires binding of DNA with a zinc metalloprotein, characterized as a zinc finger Cys-X2-5-Cys2-15-Cys/His-X2-4-Cys/His, with Cys=cysteine and X=a variable residue, each of subscripted numbers (Chinami, 1996). Zinc finger domains have been suggested as therapeutic targets for HSV (Beerheide, 1999; Scozzafava, 2002).
A recent review by AJ Kesel (2003) depicted the zinc finger domains (or motifs) in HIV-1. Similar motifs are reported in various viruses such as Picornaviridae (poliovirus, human coxsackievirus, hepatitis A virus), Flaviviridae (yellow fever virus, Dengue virus, West Nile virus, Kunjin virus, St. Louis encephalitis virus, hepatitis C virus), Togaviridae (rubella virus), Coronaviridae (human coronavirus, human SARS-associated coronavirus), Rhabdoviridae (rabies virus), Paramyxoviridae (human parainfluenza virus, measles virus, human respiratory syncytial virus), Filoviridae (Marburg virus, Ebola virus), Bornaviridae (Borna disease virus), Bunyaviridae (Hantaan virus), Arenaviridae (Lassa virus), Reoviridae (human rotavirus) and Retroviridae (HTLV-1, HIV-1, HIV-2).
Malaria
Trypanosomatids, the causative parasite of malaria, contains a single mitochondrion containing kinetoplast DNA—a unique extrachromosomal DNA network. Two short sequences of these DNA minicircles of this network are conserved in all trypanosomatid species. A protein containing five CCHC-type zinc finger domains, designated universal minicircle sequence-binding protein (UMSBP), mediates DNA-binding activity and protein oligomerization throughout the trypanosomatid cell cycle. Importantly, this activity has been shown to be redox-sensitive (oxidation state dependent). Reported to regulate UMSBP activity through a redox-based biological switch (Sela, 2008), platinum(IV) coordination to the zinc finger domain could impair trypanosomatids and be useful in the treatment of malaria.
Tat Proteins
Trans-activator of transcription proteins (Tat) are encoded by lentiviruses. Reported as early as 1988 (Frankel), Tat monomers form metal-linked dimers with metal ions through cysteine-rich regions, coordinating with either cadmium(II) or zinc(II). The cysteine-rich region of Tat proteins does not define a zinc-finger motif, per se, but five of the seven cysteine residues have been identified to coordinate two zinc(II) ions in a zinc-binding site (Huang and Wang, 1996). Coordination of zinc(II) ions appears to be critical to Tat transcriptional functions, as the Monoclonal Antibody MAb 5A4, which recognizes only the zinc-coordinated epitope, inhibited the trans-activation of the HIV long terminal repeat (LTR) in HeLa-CD4-LTR/beta-gal cells induced by treatment with the recombinant Tat protein (Misumi, 2004). Tat proteins interact with various cell receptors, including N-methyl-d-aspartate (NMDA) receptors. Its potentiating effects are associated with zinc(II) ions (Chandra et al., 2005). The sequence of a representative HIV-1 Tat protein below highlights cysteine residues of this region. Pt(IV) compounds are proposed to interact with the cysteine-rich region of Tat proteins, thus altering their activities.
Tat Protein, NCBI Accession Number AF224507.1:
SEQ ID NO 16:mepvdprlep wkhpgsqpkt pctkcyckkcclhcqvcfmt kglgisygrk krrqrrrapq dnknhqvsls kqptsrargd ptgqeeskek veketvvdpv t
Tat proteins are produced by HIV-1 infected cells, and are sufficient to elicit detrimental pathological responses. Tat increases proliferation, sensitizes cells to apoptosis, and changes the conformation of Sp1, affecting its ability to bind to its cognate DNA sequence and to retain its zinc (Seve et al., 1999). Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence (Egele et al., 2008). Importantly, holo-Tat (Tat protein coordinated to zinc(II) ions) appears to differ from apo-Tat (without zinc ions) in this activity (Egele, et al., 2008). The MAb 5A4 also inhibits apoptosis of Jurkat cells induced by treatment with the released native-Tat-protein-containing supernatant from the culture of HIV-1 (JRFL)-infected cells (Misumi et al., 2004).
Tat proteins have been shown to directly cause neurological damage or to activate microglial cells (King et al., 2006). They transport across the blood-brain-barrier and uniquely transport along neuronal pathways (Banks et al., 2005; Li et al., 2009). The integrity of the blood-brain-barrier itself is markedly affected by Tat proteins through the expression and distribution of specific tight junction proteins in brain endothelium (Andras et al., 2003), thus contributing to HIV trafficking into the central nervous system. Further relationships between Tat-caveolin signaling pathways (Zhong et al., 2008) and the localization of Pt(IV) compounds to caveolae may present targeting advantages for Pt(IV) in Tat regulation. Current HIV therapeutics do not affect Tat proteins after the lentivirus proviral DNA has integrated into host cells, such that Tat proteins continue to cause pathological damage independent of viral load.
Tat proteins have also been proposed to be useful in the development of vaccines (Campbell and Loret, 2009; Caputo et al., 2009). Their cell-penetrating capacities serve well as transporters, including cargoes of peptides, proteins, drugs, oligonucleotides, imaging agents, nanoparticles, micelles and liposomes (Wadia and Dowdy, 2005; Rapoport and Lorberboum-Galski, 2009). Whether the coordination of Tat proteins to metals such as Pt(IV) would render these transporters more useful—and less pathogenic—is likely. Since Pt(IV) would compete with biologically available metal cations for coordination, and prove to be more thermodynamically (or kinetically) stable, this could reduce detrimental effects of Tat proteins in their use as vectors and perhaps serve advantageously to stabilize protein conformations, even protecting from degradation. In this regard, coordination with zinc(II) cations deters Tat oxidation (Egele et al., 2008).
Tristetraprolin
Tristetraprolin (TTP), also known as TIS11, Nup475 or zinc finger protein 36 homolog (ZFP36), is a protein containing zinc binding domains of the CCCH type, each coordinating one zinc atom. The tandem zinc fingers consist of CX8CX5CX3H sequences (Genbank accession number X02611), where C is cysteine and X refers to variable amino acids with the subscript designating numbers of these. Alignments of these tandem zinc binding domains of all vertebrate members of the subclass are highly conserved, reflecting the importance of the domain. Tristetraprolin is important to macrophages as an intracellular regulator of tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) through message (m)RNA-stabilizing interactions. TTP knockout mice overproduce TNF-alpha. Mutation of a single cysteine residue to an arginine in either zinc finger completely abolished mRNA-destabilizing activities in TTP, suggesting that the zinc finger domain is critical to these activities (Lai, 2000). Coordination of TTP to platinum(IV) may be useful to alter regulation of both TNF-alpha and GM-CSF from immune cells. TTP has also been suggested as a therapeutic target for HPV-induced cervical cancer (Gallouzi and Di Marco, 2009).
Immunological Contributions
Viral proteins containing zinc-finger domains have been noted to inhibit antigen presentation pathways in host organisms. Members of both gamma-2 herpesviruses and poxviruses have been shown to downregulate major histocompatability complex (MHC) class I (Fruh, 2002). In addition, other cell surface molecules involved in immune recognition by thymus (T-)cells and Natural Killer cells are downregulated. Homologous K3 and K5 genes of Kaposi's sarcoma-associated virus inhibited antigen presentation and decreased cell surface expression of HLA class I antigens (Stevenson, 2000). The proposed downregulations may be associated with K3 and K5 genes encoding zinc-domain-containing proteins from viral open reading frames (ORFs), affecting ubiquitin ligases that regulate the intracellular transport of transmembrane proteins through ubiquitination (Fruh, 2002). The association of Pt(IV) compounds with zinc binding domains of these proteins may prevent such viral subversions of the immune system, hence facilitating antigen presentation of the adaptive immune response and Natural Killer cell activities of the innate immune response.
Virus-like particles (VLP) are an effective type of protein subunit vaccine that mimic viral structure in the absence of the viral RNA or DNA genome. The capsid proteins of enveloped viruses, in particular, have been widely used for this purpose. VLPs present antigens causing both B-cell and T-cell immune responses (Paliard, 2000; Murata, 2003). The VLP-approach has been successfully evaluated as vaccines and adjuvants (Sedlick, 1997; Sugrue, 1997; Tacket, 2003; Warfield, 2003; Pushko, 2005; Kang, 2009); as carriers for presenting foreign epitopes (Sadeyen, 2003; Saini and Vrati, 2003); for the chemical conjugation of peptides (Kang, 2009); and for the delivery of antigenic molecules (Storni, 2004; Alvarez-Lajonchere, 2006). VLPs may induce potent immunity by continuously priming dendritic cells (Wingard, 2008). Immunization of mice with a semipurified recombinant capsid protein from Dengue-2 Virus (produced in E. coli) has been reported to confer protection against challenge of the same Dengue-2 virus (Amexis and Young, 2006).
Exposure to nucleocapsid proteins for antigen presentation may enhance immune responses (Lazo, 2010). Evidence of this include a corresponding virus humoral response increased production of interferon-gamma by spleen cells, induced dendritic cell maturation and secretion of cytokines stimulating CD4+ and CD8+ T-cells (Shresta, 2004). Additionally, the uptake of VLPs by dendritic cells may polarize cells toward a cytotoxic response. Oligodeoxynucleotides as adjuvants are capable of enhancing cell-mediated immunity; oligodeoxynucleotides packaged into VLP's are particularly effective (Riedl, 2002; Gil, 2009). In addition to neutralizing antibodies, an antiviral response achieved utilizing VLPs might be produced through cell-mediated cytotoxicity (Sedlik, 1997; Greenstone, 1998) or complement activation. Long term humoral and cellular protective responses have been demonstrated in baboons (Jeong, 2004) and chimpanzees (Elmowalid, 2007) immunized with Hepatitis-C VLPs containing either structural proteins or oligodeoxynucleotides. Data evidence production of nucleotide-defective and nucleotide-deficient virion particles with morphologically altered phenotypes upon exposure of HIV-infected host cells to platinum(IV) compound, FX101. Formation of these virus-like particles may account for observed long term virus control in FIV-infected cats.
A20 Proteins
Cytoplasmic A20 is a zinc finger protein with ubiquitin-modifying activity. Ubiquitin, a small protein rich in lysine residues, is covalently attached to numerous proteins by ligases. Ubiquitylation affects protein stability, function, and intracellular localization; sorting to the proteosome by ubiquitin results in degradation.
Human A20 is a 790-residue protein comprising seven zinc fingers of the Cys2/Cys2 type at the C-terminus. Importantly, A20 is a negative regulator of nuclear factor kappa beta (NF-κB) signaling and activator protein-1; in dendritic cells, down-regulation of A20 results in dendritic cells with enhanced T-cell stimulatory capacity (Breckpot, 2009). A20 is also a negative regulator of the toll-like receptor and tumor necrosis factor (TNF) receptor signaling pathways, controlling the maturation, cytokine production and immunostimulatory potency of dendritic cells (Song, 2008). A20 expression is up-regulated by either TNF-alpha or lipopolysaccharide (LPS) stimulation. The effect of Pt(IV) compounds on proteins such as A20 may impact associated functions.
Prion Proteins
Transmissible Spongiform Encephalopathies (TSE) are a unique class of neurodegenerative diseases where the disease agent is a prion protein. Prion protein binds copper in 4 tandem repeats of PHGGGWGQ (SEQ ID NO: 17); adjacent histidines (H96 and H111) may also bind copper. In addition to copper, other metals have been associated with Prion protein—notably zinc (Zn2+). Zinc is also the only metal other than copper that induces PrP endocytosis and inhibits fibril formation in synthetic prions (Perera and Hooper, 2001; Walter, 2007).
Zinc Fingers and DNA Regulation
The regulation of DNA by zinc fingers is well known. Whether Pt(IV) compounds could interact with zinc fingers within the nucleus would depend upon their transport into the compartment and selectivity requirements. Nonspecific interactions could be overcome by association with specific proteins or oligonucleotides. Cobalt(III) oligonucleotides, for example, are reported to selectively target Snail family zinc transcription factors (Harney, 2009).
The ubiquitous transcription protein Sp1 contains a zinc finger domain of the CysCysHisHis (CCHH) type, binding to the GC box elements of DNA. Sp1 interacts with numerous factors (Deniaud, 2009). It is possible that certain Pt(IV) compounds could interact with such zinc domains, especially if positively charged. The cellular role of CCHH-type zinc domains in affecting protein interactions is proposed to be much greater than their role in DNA binding (Brayer, 2008), and thus possibly self-limiting. The number of zinc finger domains per protein would also contribute to an optimization of the therapeutic use (Koellensperger, 2007).